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Dysfunction of Nrf-2 in CF epithelia leads to excess intracellular H2O2 and inflammatory cytokine production.

Chen J, Kinter M, Shank S, Cotton C, Kelley TJ, Ziady AG - PLoS ONE (2008)

Bottom Line: We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized.The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2.We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio, USA.

ABSTRACT
Cystic fibrosis is characterized by recurring pulmonary exacerbations that lead to the deterioration of lung function and eventual lung failure. Excessive inflammatory responses by airway epithelia have been linked to the overproduction of the inflammatory cytokine IL-6 and IL-8. The mechanism by which this occurs is not fully understood, but normal IL-1beta mediated activation of the production of these cytokines occurs via H2O2 dependent signaling. Therefore, we speculated that CFTR dysfunction causes alterations in the regulation of steady state H2O2. We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized. Increases in H2O2 heavily contributed to the excessive IL-6 and IL-8 production in CF epithelia. Proteomic analysis of three in vitro and two in vivo models revealed a decrease in antioxidant proteins that regulate H2O2 processing, by > or =2 fold in CF vs. matched normal controls. When cells are stimulated, differential expression in CF versus normal is enhanced; corresponding to an increase in H2O2 mediated production of IL-6 and IL-8. The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2. Inhibition of CFTR function in normal cells produced this phenotype, while N-acetyl cysteine, selenium, an activator of Nrf-2, and the overexpression of Nrf-2 all normalized H2O2 processing and decreased IL-6 and IL-8 to normal levels, in CF cells. We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8. Treatment with antioxidants can ameliorate exaggerated cytokine production without affecting normal responses.

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Expression of antioxidant proteins is altered in cystic fibrosis.Non-stimulated models of CF are compared by 2-D gel analysis. For in vitro studies, 16HBEo− (sense and antisense (AS)) and 9HTEo− (pCEP and pCEP-R) cell line pairs are grown to 80% confluence on 5 different occasions, homogenized, and the homogenates equally loaded onto 2-D gels. For in vivo studies, 5 sets of excised nasal epithelial or whole lung tissues from 8 week old wildtype mice or their CF litter mates were harvested, homogenized and total protein analyzed. Gels were scanned by densitometer, averaged and a comparative densities calculated (see Tables 1 and 2). Representative 2-D gel bands from CF and normal matched pairs are shown for each model. Blue protein names indicate a decrease, while red names indicate an increase in expression in CF.
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pone-0003367-g003: Expression of antioxidant proteins is altered in cystic fibrosis.Non-stimulated models of CF are compared by 2-D gel analysis. For in vitro studies, 16HBEo− (sense and antisense (AS)) and 9HTEo− (pCEP and pCEP-R) cell line pairs are grown to 80% confluence on 5 different occasions, homogenized, and the homogenates equally loaded onto 2-D gels. For in vivo studies, 5 sets of excised nasal epithelial or whole lung tissues from 8 week old wildtype mice or their CF litter mates were harvested, homogenized and total protein analyzed. Gels were scanned by densitometer, averaged and a comparative densities calculated (see Tables 1 and 2). Representative 2-D gel bands from CF and normal matched pairs are shown for each model. Blue protein names indicate a decrease, while red names indicate an increase in expression in CF.

Mentions: To investigate the mechanism of the increase of H2O2 in CF cells, we used a proteomic approach to analyze relevant redox proteins. We examined 2-D gels equally loaded with whole cell protein from the cultured CF model 9HTEo− and 16HBEo− cell pairs. Comparisons of five gel maps for untreated normal and CF cells indicated the differential expression by 2 fold or higher of the key redox proteins thioredoxin 1 (TRX-1), Mn superoxide dismutase (SOD2), glutathione-S-transferase pi (GST-pi), peroxiredoxin (PRDX) 6, TRX dependent peroxide reductase (PRDX-1), and catalase (Figure 3, Table 1). Spots were identified using LC-MS to obtain CID spectra with sequence information (example CID spectra, Figure S1a–f), that were each matched to the human protein database (NCBI) with greater than 95% confidence (Table S1). Our protein identifications are based on a sequence coverage range average of 28%–67% (Table S1). This translates to 4–24 different tryptic peptides whose sequence match entries in public databases for each protein, further increasing our confidence in our identifications. Importantly, for each of our bands of interest we only detected tryptic peptides belonging to an individual protein. The lack of detection of any peptides from secondary proteins indicated that the level of any contamination (>10 fold less abundant) would not affect our accuracy of quantitation, and that the direct comparison of Coomassie stained 2D gel bands for this set of proteins was valid.


Dysfunction of Nrf-2 in CF epithelia leads to excess intracellular H2O2 and inflammatory cytokine production.

Chen J, Kinter M, Shank S, Cotton C, Kelley TJ, Ziady AG - PLoS ONE (2008)

Expression of antioxidant proteins is altered in cystic fibrosis.Non-stimulated models of CF are compared by 2-D gel analysis. For in vitro studies, 16HBEo− (sense and antisense (AS)) and 9HTEo− (pCEP and pCEP-R) cell line pairs are grown to 80% confluence on 5 different occasions, homogenized, and the homogenates equally loaded onto 2-D gels. For in vivo studies, 5 sets of excised nasal epithelial or whole lung tissues from 8 week old wildtype mice or their CF litter mates were harvested, homogenized and total protein analyzed. Gels were scanned by densitometer, averaged and a comparative densities calculated (see Tables 1 and 2). Representative 2-D gel bands from CF and normal matched pairs are shown for each model. Blue protein names indicate a decrease, while red names indicate an increase in expression in CF.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2563038&req=5

pone-0003367-g003: Expression of antioxidant proteins is altered in cystic fibrosis.Non-stimulated models of CF are compared by 2-D gel analysis. For in vitro studies, 16HBEo− (sense and antisense (AS)) and 9HTEo− (pCEP and pCEP-R) cell line pairs are grown to 80% confluence on 5 different occasions, homogenized, and the homogenates equally loaded onto 2-D gels. For in vivo studies, 5 sets of excised nasal epithelial or whole lung tissues from 8 week old wildtype mice or their CF litter mates were harvested, homogenized and total protein analyzed. Gels were scanned by densitometer, averaged and a comparative densities calculated (see Tables 1 and 2). Representative 2-D gel bands from CF and normal matched pairs are shown for each model. Blue protein names indicate a decrease, while red names indicate an increase in expression in CF.
Mentions: To investigate the mechanism of the increase of H2O2 in CF cells, we used a proteomic approach to analyze relevant redox proteins. We examined 2-D gels equally loaded with whole cell protein from the cultured CF model 9HTEo− and 16HBEo− cell pairs. Comparisons of five gel maps for untreated normal and CF cells indicated the differential expression by 2 fold or higher of the key redox proteins thioredoxin 1 (TRX-1), Mn superoxide dismutase (SOD2), glutathione-S-transferase pi (GST-pi), peroxiredoxin (PRDX) 6, TRX dependent peroxide reductase (PRDX-1), and catalase (Figure 3, Table 1). Spots were identified using LC-MS to obtain CID spectra with sequence information (example CID spectra, Figure S1a–f), that were each matched to the human protein database (NCBI) with greater than 95% confidence (Table S1). Our protein identifications are based on a sequence coverage range average of 28%–67% (Table S1). This translates to 4–24 different tryptic peptides whose sequence match entries in public databases for each protein, further increasing our confidence in our identifications. Importantly, for each of our bands of interest we only detected tryptic peptides belonging to an individual protein. The lack of detection of any peptides from secondary proteins indicated that the level of any contamination (>10 fold less abundant) would not affect our accuracy of quantitation, and that the direct comparison of Coomassie stained 2D gel bands for this set of proteins was valid.

Bottom Line: We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized.The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2.We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio, USA.

ABSTRACT
Cystic fibrosis is characterized by recurring pulmonary exacerbations that lead to the deterioration of lung function and eventual lung failure. Excessive inflammatory responses by airway epithelia have been linked to the overproduction of the inflammatory cytokine IL-6 and IL-8. The mechanism by which this occurs is not fully understood, but normal IL-1beta mediated activation of the production of these cytokines occurs via H2O2 dependent signaling. Therefore, we speculated that CFTR dysfunction causes alterations in the regulation of steady state H2O2. We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized. Increases in H2O2 heavily contributed to the excessive IL-6 and IL-8 production in CF epithelia. Proteomic analysis of three in vitro and two in vivo models revealed a decrease in antioxidant proteins that regulate H2O2 processing, by > or =2 fold in CF vs. matched normal controls. When cells are stimulated, differential expression in CF versus normal is enhanced; corresponding to an increase in H2O2 mediated production of IL-6 and IL-8. The cause of this redox imbalance is a decrease by approximately 70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2. Inhibition of CFTR function in normal cells produced this phenotype, while N-acetyl cysteine, selenium, an activator of Nrf-2, and the overexpression of Nrf-2 all normalized H2O2 processing and decreased IL-6 and IL-8 to normal levels, in CF cells. We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8. Treatment with antioxidants can ameliorate exaggerated cytokine production without affecting normal responses.

Show MeSH
Related in: MedlinePlus